Project Summary Inhibitors of cyclin-dependent kinases CDK4 and CDK6 have been approved for treatment of luminal-type estrogen receptor (ER)-positive breast cancers. Unfortunately, a large proportion of patients with breast cancer develops resistance to CDK4/6 inhibition. In Aim 1, we will test our hypothesis that CDK4/6 resistant breast cancer cells become dependent on hyperactivation of the cyclin-dependent kinase CDK2 for proliferation. Consequently, we hypothesize that inhibition of CDK2 in CDK4/6 inhibitor-resistant cancer cells would block their proliferation. We further hypothesize that combined inhibition of CDK4/6 and CDK2 would have a synergistic effect, and might prevent the development of resistant disease. Currently, a major limitation in studying the role of CDK2 is the absence of CDK2-specific inhibitors. To overcome this limitation, our laboratory has applied the ?analog-sensitive? kinase approach, which allows us to specifically, potently and reversibly inhibit CDK2 using a compound that does not inhibit any other kinases. In Aim 1, we will use the analog-sensitive approach to test the impact of CDK2 inhibition on proliferation of CDK4/6-inhibitor resistant tumors in vivo. We will also compare side-by-side the effects of potent CDK2 inhibition using our system, versus CDK2 inhibitors that are currently in clinical trials. In an effort to assess the role of CDK2 in the development of CDK4/6 resistance in the clinic, we will obtain 120 baseline biopsies from patients starting CDK4/6 inhibitor treatment to obtain 60 paired biopsies at baseline and when resistance develops. These biopsies will be interrogated for CDK2 activation status, and we will develop new approaches to gauge CDK2 activity in the clinical setting. In Aim 2, we will extend our investigations to triple negative breast cancer (TNBC). In contrast to luminal-type breast cancers, TNBC is intrinsically resistant to CDK4/6 inhibition. Nonetheless, we have observed that a significant fraction of human TNBC cell lines critically requires CDK4/6 for proliferation. Our preliminary data indicate that CDK4/6 inhibitors become sequestered into TNBC cell lysosomes, thereby blocking the inhibitors? therapeutic effect. Importantly, we found that treatment of TNBC cells with compounds that inhibit lysosomal acidification, such as chloroquine, reverses the sequestration and renders TNBC cells sensitive to CDK4/6 inhibitor treatment. We also identified a new CDK4/6 inhibitor compound that on its own inhibits proliferation of TNBC cells. We will test the utility of combining CDK4/6 inhibitors with chloroquine for treatment of TNBC, using patient-derived xenografts, as well as short-term cultures of cells isolated directly from human tumors. We will also use these systems to evaluate the efficacy of the novel CDK4/6 inhibitor described above. We will conduct a phase I/II study of palbociclib and chloroquine to test the hypothesis that the addition of chloroquine can circumvent lysosomal sequestration, and patients in this trial will undergo paired biopsies to assess CDK4/6 inhibitor sequestration. The expected overall impact of this proposal is that it may provide a highly effective therapeutic strategy for overcoming acquired resistance to CDK4/6 inhibitors, and may extend the benefits of anti-CDK4/6 therapy to patients with TNBC.